Lens fiber cell differentiation involves a coordinated change in gene expression. A number of genes including Pax6, FoxE3 and E-cadherin are expressed in lens epithelial cells but down-regulated in lens fiber cells. Conversely many other genes including Aquaporin0, 2- and 3-crystallins and lens-specific beaded filament proteins CP49 and filensin are not expressed in lens epithelial cells but are turned on in lens fiber cells. In recent years it has become increasingly clear that epigenetic modification (reversible covalent modification of DNA or histone proteins) of chromosomal DNA plays a major role in gene regulation. Among the best studied epigenetic modifications is methylation of cytosine in CpG dinucleotides in DNA. Methylation of DNA in mammalian cells is accomplished by Dnmt1, which is responsible for maintaining epigenetic methylation through cell divisions, and Dnmt3a and Dnmt3b, which are responsible for creating de novo methylation changes during development. Promoter DNA methylation is associated with transcriptional repression of genes. Despite the wealth of knowledge of transcription factors involved in lens development, very little is known about the epigenetic regulation of lens fiber cell differentiation. Recent evidence suggests that Dnmt1 and Dnmt3 activity are specifically required for lens development in zebrafish. We hypothesize that the balance between promoter methylation and demethylation is required for proper lens fiber cell differentiation, and that this balance will require the activity of both maintenance and de novo methylation activity. We propose that maintenance methylation will be required to prevent the expression of genes associated with fiber cell differentiation in the lens epithelium and that de novo methylation will be required to repress the expression of lens epithelial genes during fiber cell differentiation. We will investigate the role of DNA methylation in fiber cell differentiation using both conditional genetic strategies in mice lacking maintenance or de novo methylases in the lens lineage. We will use histological, immunological and high throughput next generation sequencing strategies to comprehensively investigate how DNA methylation influences lens fiber cell differentiation. PUBLIC HEALTH RELEVANCE: Recent experiments demonstrating that epigenetic reprogramming can convert differentiated cell types into pluripotent stem cells makes clear the critical importance for understanding the epigenetic regulation of the differentiated phenotype. We propose that the lens represents a unique opportunity to understand how epigenetic DNA methylation regulates differentiation. This understanding will be important not only for lens development, but for a global understanding of how to manipulate differentiated states, which is critical for the generation of patient-specific stem cells in medicine.